Search

Sensory adaptation is the process whereby brain circuits adjust neuronal activity in response to redundant sensory stimuli. Although sensory adaptation has been extensively studied for individual neurons on timescales of tens of milliseconds to a few seconds, little is known about it over longer timescales or at the population level. We investigated population-level adaptation in the barrel field of the mouse somatosensory cortex (S1BF) using in vivo two-photon calcium imaging and Neuropixels recordings in awake mice. Among stimulus-responsive neurons, we found both adapting and facilitating neurons, which decreased or increased their firing, respectively, with repetitive whisker stimulation. The former outnumbered the latter by 2:1 in layers 2/3 and 4; hence, the overall population response of mouse S1BF was slightly adapting. We also discovered that population adaptation to one stimulus frequency (5 Hz) does not necessarily generalize to a different frequency (12.5 Hz). Moreover, responses of individual neurons to repeated rounds of stimulation over tens of minutes were strikingly heterogeneous and stochastic, such that their adapting or facilitating response profiles were not stable across time. Such representational drift was particularly striking when recording longitudinally across 8-9 days, as adaptation profiles of most whisker-responsive neurons changed drastically from one day to the next. Remarkably, repeated exposure to a familiar stimulus paradoxically shifted the population away from strong adaptation and toward facilitation. Thus, the adapting vs. facilitating response profile of S1BF neurons is not a fixed property of neurons but rather a highly dynamic feature that is shaped by sensory experience across days.

BackgroundExaggerated responses to sensory stimuli, a hallmark of Fragile X syndrome (FXS), contribute to anxiety and learning challenges. Sensory hypersensitivity is recapitulated in the Fmr1 knockout (KO) mouse model of FXS. Recent studies in Fmr1 KO mice have demonstrated differences in activity of cortical interneurons and a delayed switch in the polarity of GABA signaling during development. Previously, we reported that blocking the chloride transporter NKCC1 with the diuretic bumetanide, could rescue synaptic circuit phenotypes in primary somatosensory cortex (S1) of Fmr1 KO mice. However, it remains unknown whether bumetanide can rescue earlier circuit phenotypes or sensory hypersensitivity in Fmr1 KO mice.MethodsWe used acute and chronic systemic administration of bumetanide in Fmr1 KO mice and performed in vivo 2-photon calcium imaging to record neuronal activity, while tracking mouse behavior with high-resolution videos.ResultsWe demonstrate that layer (L) 2/3 pyramidal neurons in S1 of Fmr1 KO mice show a higher frequency of synchronous events at postnatal day (P) 6 compared to wild-type controls. This was reversed by acute administration of bumetanide. Furthermore, chronic bumetanide treatment (P5-P14) restored S1 circuit differences in Fmr1 KO mice, including reduced neuronal adaptation to repetitive whisker stimulation, and ameliorated tactile defensiveness. Bumetanide treatment also rectified the reduced feedforward inhibition of L2/3 neurons in S1 and boosted the circuit participation of parvalbumin interneurons.ConclusionsThis further supports the notion that synaptic, circuit, and sensory behavioral phenotypes in Fmr1 KO can be mitigated by inhibitors of NKCC1, such as the FDA-approved diuretic bumetanide.

SIGNIFICANCE: Pain comprises a complex interaction between motor action and somatosensation that is dependent on dynamic interactions between the brain and spinal cord. This makes understanding pain particularly challenging as it involves rich interactions between many circuits (e.g., neural and vascular) and signaling cascades throughout the body. As such, experimentation on a single region may lead to an incomplete and potentially incorrect understanding of crucial underlying mechanisms. AIM: We aimed to develop and validate tools to enable detailed and extended observation of neural and vascular activity in the brain and spinal cord. The first key set of innovations was targeted to developing novel imaging hardware that addresses the many challenges of multisite imaging. The second key set of innovations was targeted to enabling bioluminescent (BL) imaging, as this approach can address limitations of fluorescent microscopy including photobleaching, phototoxicity, and decreased resolution due to scattering of excitation signals. APPROACH: We designed 3D-printed brain and spinal cord implants to enable effective surgical implantations and optical access with wearable miniscopes or an open window (e.g., for one- or two-photon microscopy or optogenetic stimulation). We also tested the viability for BL imaging and developed a novel modified miniscope optimized for these signals (BLmini). RESULTS: We describe universal implants for acute and chronic simultaneous brain-spinal cord imaging and optical stimulation. We further describe successful imaging of BL signals in both foci and a new miniscope, the BLmini, which has reduced weight, cost, and form-factor relative to standard wearable miniscopes. CONCLUSIONS: The combination of 3D-printed implants, advanced imaging tools, and bioluminescence imaging techniques offers a coalition of methods for understanding spinal cord-brain interactions. Our work has the potential for use in future research into neuropathic pain and other sensory disorders and motor behavior.

Electron‐rich symmetric pull‐push‐pull molecules can act as efficient two‐photon absorbing units. When these chromophores are bonded directly to photo‐switchable molecules they can function as antenna systems to indirectly induce photochemical transformations in isomerizable groups after energy transfer. Recently, we developed an antenna–molecular switch system based on a pyrrolo–pyrrole two‐photon active chromophore. When this antenna section is functionalized with symmetrically situated azo‐sections as N,N’‐pyrrolic‐substituents, these azo molecular switches can be efficiently transformed from their E to their Z isomers after non‐linear light absorption by the antenna, followed by indirect excitation of the azo‐sections. In this contribution we present a case‐study of one of these systems through femtosecond‐resolved fluorescence to observe the dynamics involved in the excitation and relaxation steps within the antenna section, as well as the energy transfer pathways. By time‐resolving the emission signals we observed that the energy transfer can occur in parallel with the relaxation within the first singlet vibronic states localized at the pyrrolo–pyrrole antenna. In fact, the antenna‐to‐azo section energy transfer shows a biphasic nature. At early times, during the relaxation within the antenna, there is an initial population of azo‐section excited molecules. In addition, after the system has evolved to the fully relaxed S1 state at the antenna section, the energy transfer has components related to thermal fluctuations which increase the couplings with the receiver states at the azo‐switches giving transfer rates of the order of 1010 s−1 at room temperature. The characterization of these relaxation and energy transfer steps, as well as the role of the solvent in these processes, gives insights for the development of future molecules with indirect two‐photon isomerization properties using this kind of pull‐push‐pull two‐photon active antenna. Due to their two‐photon reactive properties, these systems can have applications in schemes where highly localized photo‐isomerization is required. [ABSTRACT FROM AUTHOR]

Despite an abundance of pharmacologic and surgical epilepsy treatments, there remain millions of patients suffering from poorly controlled seizures. One approach to closing this treatment gap may be found through a deeper mechanistic understanding of the network alterations that underly this aberrant activity. Functional optical imaging in vertebrate models provides powerful advantages to this end, enabling the spatiotemporal acquisition of individual neuron activity patterns across multiple seizures. This coupled with the advent of genetically encoded indicators, be them for specific ions, neurotransmitters or voltage, grants researchers unparalleled access to the intact nervous system. Here, we will review how in vivo functional optical imaging in various vertebrate seizure models has advanced our knowledge of seizure dynamics, principally seizure initiation, propagation and termination. [ABSTRACT FROM AUTHOR]

Calcium imaging, especially two-photon imaging, has become essential in neuroscience for studying neuronal and astrocytic activity under in vivo and in vitro conditions. Current advances in the development of calcium sensors as well as imaging hardware enable high-frequency measurements of calcium signals in hundreds of cells simultaneously. The analysis of these large datasets requires special tools and usually a certain level of programming experience. Despite advancements in calcium imaging analysis software development, significant gaps remain, particularly for data acquired at a high sampling rate that would allow for the spectral analysis of calcium signals. The FluoAnalysis MATLAB toolbox addresses these gaps by offering a comprehensive solution for analyzing simultaneously measured calcium imaging and electrophysiological data. It features both GUI-based and command-line approaches, emphasizing frequency domain analysis to reveal network-level oscillatory signals linked to single-cell activity. In addition, the toolbox puts special emphasis on differentiating between astrocytes and neurons, revealing the interactions between the network activity of the two major cell types of the brain. It facilitates a streamlined workflow for data loading, ROI identification, cell classification, fluorescence intensity calculation, spectral analysis, and report generation, supporting both manual and automated high-throughput analysis. This versatile platform enables the comprehensive analysis of large imaging datasets. In conclusion, the FluoAnalysis MATLAB toolbox provides a robust and versatile platform for the integrated analysis of calcium imaging and electrophysiological data, supporting diverse neuroscience research applications. [ABSTRACT FROM AUTHOR]

Significance: Genetically encoded voltage indicators (GEVIs) are a valuable tool for studying neural circuits in vivo, but the relative merits and limitations of onephoton (1P) versus two-photon (2P) voltage imaging are not well characterized. Aim: We consider the optical and biophysical constraints particular to 1P and 2P voltage imaging and compare the imaging properties of commonly used GEVIs under 1P and 2P excitation. Approach: We measure the brightness and voltage sensitivity of voltage indicators from commonly used classes under 1P and 2P illumination. We also measure the decrease in fluorescence as a function of depth in the mouse brain. We develop a simple model of the number of measurable cells as a function of reporter properties, imaging parameters, and desired signal-to-noise ratio (SNR). We then discuss how the performance of voltage imaging would be affected by sensor improvements and by recently introduced advanced imaging modalities. Results: Compared with 1P excitation, 2P excitation requires ~104-fold more illumination power per cell to produce similar photon count rates. For voltage imaging with JEDI-2P in the mouse cortex with a target SNR of 10 (spike height to baseline shot noise), a measurement bandwidth of 1 kHz, a thermally limited laser power of 200 mW, and an imaging depth of >300 µm, 2P voltage imaging using an 80-MHz source can record from no more than ~12 neurons simultaneously. Conclusions: Due to the stringent photon-count requirements of voltage imaging and the modest voltage sensitivity of existing reporters, 2P voltage imaging in vivo faces a stringent tradeoff between shot noise and tissue photodamage. 2P imaging of hundreds of neurons with high SNR at a depth of >300 µm will require either major improvements in 2P GEVIs or qualitatively new approaches to imaging. [ABSTRACT FROM AUTHOR]

Changes in the function of inhibitory interneurons (INs) during cortical development could contribute to the pathophysiology of neurodevelopmental disorders. Using all-optical in vivo approaches, we find that parvalbumin (PV) INs and their immature precursors are hypoactive and transiently decoupled from excitatory neurons in postnatal mouse somatosensory cortex (S1) of Fmr1 KO mice, a model of fragile X syndrome (FXS). This leads to a loss of parvalbumin INs (PV-INs) in both mice and humans with FXS. Increasing the activity of future PV-INs in neonatal Fmr1 KO mice restores PV-IN density and ameliorates transcriptional dysregulation in S1, but not circuit dysfunction. Critically, administering an allosteric modulator of Kv3.1 channels after the S1 critical period does rescue circuit dynamics and tactile defensiveness. Symptoms in FXS and related disorders could be mitigated by targeting PV-INs.

Discriminating between temporal features in sensory stimuli is critical to complex behavior and decision-making. However, how sensory cortical circuit mechanisms contribute to discrimination between subsecond temporal components in sensory events is unclear. To elucidate the mechanistic underpinnings of timing in primary visual cortex (V1), we recorded from V1 using two-photon calcium imaging in awake-behaving mice performing a go/no-go discrimination timing task, which was composed of patterns of subsecond audiovisual stimuli. In both conditions, activity during the early stimulus period was temporally coordinated with the preferred stimulus. However, while network activity increased in the preferred condition, network activity was increasingly suppressed in the nonpreferred condition over the stimulus period. Multiple levels of analyses suggest that discrimination between subsecond intervals that are contained in rhythmic patterns can be accomplished by local neural dynamics in V1.

Excitation in MRI is traditionally done at the Larmor frequency, where the energy of each radiofrequency photon corresponds to the energy difference between two spin states. However, if multiple radiofrequencies are employed, then multiphoton excitation can also occur when the sum or difference of multiple photon frequencies equals the Larmor frequency. Although multiphoton excitation has been known since the early days of NMR, it has been relatively unexplored in MRI. In this work, equations and principles for multiphoton selective RF pulse design in imaging are presented and experimentally demonstrated. In particular, the case where there are radiofrequency fields in both the traditional xy-direction and non-traditional z-direction is considered. To produce the z-direction radiofrequency field, an additional uniform coil was added to a clinical MRI scanner. Using this coil, two-photon slice-selective pulses were designed to be equivalent to traditional pulses, producing similar excitation, slice profiles, and in vivo images. Being the result of a combination of multiple radiofrequency fields instead of just one, two-photon pulses have more flexibility in how their parameters can be changed. Although individual multiphoton excitations are less efficient than their traditional counterparts, when the z-direction radiofrequency field is spatially non-uniform, multiple multiphoton resonances can be simultaneously used at different locations to produce simultaneous multislice excitation with the same pulse duration but less tissue heating than a naive implementation. In particular, non-uniform z-direction radiofrequency fields with negligible added tissue heating provided by oscillating the MRI scanners gradient fields at kilohertz frequencies were used to excite multiple slices simultaneously with less high-frequency xy-direction radiofrequency power. For an example three-slice excitation, we achieve half the xy-direction radiofrequency power compared to the naïve approach of adding three single-slice pulses. For conventional or unconventional applications, multiphoton excitation may be of interest when designing new MRI systems.

An effective, GFP‐inspired fluorescent Zn2+ sensor is developed for two–photon microscopy and related biological application that features an 8‐methoxyquinoline moiety. Excellent photophysical characteristics including a 37‐fold fluorescence enhancement with excitation and emission maxima at 440 nm and 505 nm, respectively, as well as a high two–photon cross–section of 73 GM at 880 nm are reported. Based on the experimental data, the relationship between the structure and properties was elucidated and explained backed up by DFT calculations, particularly the observed PeT phenomenon for the turn–on process. Biological validation and detailed experimental and theoretical characterization of the free and the zinc–bound compounds are presented. [ABSTRACT FROM AUTHOR]

As a unique type of supramolecular self‐assemblies, crystalline host‐guest aggregates have attracted extensive interests in multiple application fields. Herein, a crystalline host‐guest aggregate LIFM‐HG1 was obtained with curcubit[8]uril as the host and carboxypyridinium salt as the guest. Single‐crystal structural analysis indicates that the presence of abundant weak interactions in LIFM‐HG1 provides a rigid environment for the guest molecule and effectively blocks the external quenchers. Spectral analysis and theoretical calculations confirm the presence of robust triplet energy levels in LIFM‐HG1. Even more impressively, the intersystem crossing channels of the guest molecules are greatly opened up after the formation of the crystalline host‐guest aggregate, resulting in a large kisc of 6.70 × 107 s−1 at room temperature for LIFM‐HG1 (which is ∼0 for pure guest), leading to fascinating multichannel (including one‐photon, two‐photon, and X‐ray) excited LPL properties. In addition, the crystalline LIFM‐HG1 has a much higher triplet state luminescence efficiency under X‐ray and two‐photon excitation than that under single‐photon excitation (AP/AF = 86.8, 44.8, 10.7 under the three circumstances, respectively). And the phosphorescent emission intensity of LIFM‐HG1 is 27.6 times higher than that of the crystalline guest under X‐ray excitation. As a result, LIFM‐HG1 shows a long afterglow retention time under both single‐ and two‐photon excitation, and an impressive afterglow retention time of 1 s under X‐ray excitation. Furthermore, the excellent lysosomal targeting and low cytotoxicity by the formation of host‐guest aggregate makes LIFM‐HG1 promising to be used as a novel lysosomal‐targeted two‐photon excited phosphorescent tracer. [ABSTRACT FROM AUTHOR]

Despite an abundance of pharmacologic and surgical epilepsy treatments, there remain millions of patients suffering from poorly controlled seizures. One approach to closing this treatment gap may be found through a deeper mechanistic understanding of the network alterations that underly this aberrant activity. Functional optical imaging in vertebrate models provides powerful advantages to this end, enabling the spatiotemporal acquisition of individual neuron activity patterns across multiple seizures. This coupled with the advent of genetically encoded indicators, be them for specific ions, neurotransmitters or voltage, grants researchers unparalleled access to the intact nervous system. Here, we will review how in vivo functional optical imaging in various vertebrate seizure models has advanced our knowledge of seizure dynamics, principally seizure initiation, propagation and termination.

The Er3+/Yb3+ co-doped Y4GeO8 crystal powders were successfully synthesized using a high-temperature solid-phase method. The crystal structure of the obtained phosphors was confirmed to be pure Y4GeO8 through X-ray diffraction (XRD) analysis. A regression equation correlating Er3+/Yb3+ doping concentrations with luminescent intensity was established based on the optimized theoretical model derived from experimental design. The optimal concentrations of Er3+ and Yb3+ under 980 nm laser excitation were determined as 7.41 and 21.34%, respectively, while under 1550 nm laser excitation, the concentrations were 2.66 and 17.42%, respectively. The fluorescence emission spectra of the up-conversion samples were measured, revealing intense green and red emissions with peaks at 542, 546, and 654 nm under 980 nm excitation, and peaks at 546, 557, and 663 nm under 1550 nm excitation. These peaks correspond to transitions from 2H11/2 to 4I15/2, 4S3/2 to 4I15/2, and 4F9/2 to 4I15/2 energy levels. The relationship between up-conversion luminescence and laser operating current for the optimal samples under 980 nm and 1550 nm was investigated, uncovering that up-conversion luminescence occurs through both two-photon and three-photon processes. A detailed analysis and discussion of the up-conversion luminescence mechanisms were conducted. Furthermore, the relationship between up-conversion fluorescence and temperature for the optimal samples was studied, revealing excellent temperature-sensing characteristics under 980 nm and 1550 nm laser excitations. The calculated illumination region coordinates for the optimal samples under 980 nm and 1550 nm wavelength excitations were (0.5558, 0.4362) and (0.5256, 0.4687), respectively. The research highlights the potential of rare-earth ion-doped up-conversion luminescent materials for diverse anti-counterfeiting applications. In particular, the Y4GeO8: Yb3+/Er3+ phosphors, incorporating a dual-excitation mechanism, enhance the security of anti-counterfeiting strategies in multifaceted scenarios. The study underscores the promising developments in this field. [ABSTRACT FROM AUTHOR]

In recent years, the development of carbon dot‐based fluorescent nanoparticles for bioimaging applications has attracted the attention of the scientific community. However, many of these systems absorb and emit in the blue–green region of the electromagnetic spectrum, limiting their application in bioimaging. Herein, the facile design and development of highly efficient two‐photon excitable red‐emissive carbon dots are reported and their high performance in bioimaging applications is demonstrated. The importance of aromatic precursors in developing red‐emissive carbon dots is demonstrated. The optimized carbon dots are highly biocompatible and nontoxic, with remarkable photostability in cells under two‐photon near‐infrared excitation. The present study points to the great potential of two‐photon excitable red‐emissive carbon dot as an efficient bioimaging agent for cellular biolabeling, long‐term and real‐time cellular imaging, and high‐resolution deep‐tissue imaging in complex biological systems. [ABSTRACT FROM AUTHOR]

Regenerative medicine, which utilizes stem cells for tissue and organ repair, holds immense promise in healthcare. A comprehensive understanding of stem cell characteristics is crucial to unlock their potential. This study explores the pivotal role of optical microscopy in advancing regenerative medicine as a potent tool for stem cell research. Advanced optical microscopy techniques enable an in‐depth examination of stem cell behavior, morphology, and functionality. The review encompasses current optical microscopy, elucidating its capabilities and constraints in stem cell imaging, while also shedding light on emerging technologies for improved stem cell visualization. Optical microscopy, complemented by techniques like fluorescence and multiphoton imaging, enhances our comprehension of stem cell dynamics. The introduction of label‐free imaging facilitates noninvasive, real‐time stem cell monitoring without external dyes or markers. By pushing the boundaries of optical microscopy, researchers reveal the intricate cellular mechanisms underpinning regenerative processes, thereby advancing more effective therapeutic strategies. The current study not only outlines the future of regenerative medicine but also underscores the pivotal role of optical microscopy in both structural and functional stem cell imaging. [ABSTRACT FROM AUTHOR]

Abstract As a unique type of supramolecular self‐assemblies, crystalline host‐guest aggregates have attracted extensive interests in multiple application fields. Herein, a crystalline host‐guest aggregate LIFM‐HG1 was obtained with curcubit[8]uril as the host and carboxypyridinium salt as the guest. Single‐crystal structural analysis indicates that the presence of abundant weak interactions in LIFM‐HG1 provides a rigid environment for the guest molecule and effectively blocks the external quenchers. Spectral analysis and theoretical calculations confirm the presence of robust triplet energy levels in LIFM‐HG1. Even more impressively, the intersystem crossing channels of the guest molecules are greatly opened up after the formation of the crystalline host‐guest aggregate, resulting in a large kisc of 6.70 × 107 s−1 at room temperature for LIFM‐HG1 (which is ∼0 for pure guest), leading to fascinating multichannel (including one‐photon, two‐photon, and X‐ray) excited LPL properties. In addition, the crystalline LIFM‐HG1 has a much higher triplet state luminescence efficiency under X‐ray and two‐photon excitation than that under single‐photon excitation (AP/AF = 86.8, 44.8, 10.7 under the three circumstances, respectively). And the phosphorescent emission intensity of LIFM‐HG1 is 27.6 times higher than that of the crystalline guest under X‐ray excitation. As a result, LIFM‐HG1 shows a long afterglow retention time under both single‐ and two‐photon excitation, and an impressive afterglow retention time of 1 s under X‐ray excitation. Furthermore, the excellent lysosomal targeting and low cytotoxicity by the formation of host‐guest aggregate makes LIFM‐HG1 promising to be used as a novel lysosomal‐targeted two‐photon excited phosphorescent tracer.

The biophysical properties of existing optogenetic tools constrain the scale, speed, and fidelity of precise optogenetic control. Here, we use structure-guided mutagenesis to engineer opsins that exhibit very high potency while retaining fast kinetics. These new opsins enable large-scale, temporally and spatially precise control of population neural activity. We extensively benchmark these new opsins against existing optogenetic tools and provide a detailed biophysical characterization of a diverse family of opsins under two-photon illumination. This establishes a resource for matching the optimal opsin to the goals and constraints of patterned optogenetics experiments. Finally, by combining these new opsins with optimized procedures for holographic photostimulation, we demonstrate the simultaneous coactivation of several hundred spatially defined neurons with a single hologram and nearly double that number by temporally interleaving holograms at fast rates. These newly engineered opsins substantially extend the capabilities of patterned illumination optogenetic paradigms for addressing neural circuits and behavior.

Calcium imaging, especially two-photon imaging, has become essential in neuroscience for studying neuronal and astrocytic activity under in vivo and in vitro conditions. Current advances in the development of calcium sensors as well as imaging hardware enable high-frequency measurements of calcium signals in hundreds of cells simultaneously. The analysis of these large datasets requires special tools and usually a certain level of programming experience. Despite advancements in calcium imaging analysis software development, significant gaps remain, particularly for data acquired at a high sampling rate that would allow for the spectral analysis of calcium signals. The FluoAnalysis MATLAB toolbox addresses these gaps by offering a comprehensive solution for analyzing simultaneously measured calcium imaging and electrophysiological data. It features both GUI-based and command-line approaches, emphasizing frequency domain analysis to reveal network-level oscillatory signals linked to single-cell activity. In addition, the toolbox puts special emphasis on differentiating between astrocytes and neurons, revealing the interactions between the network activity of the two major cell types of the brain. It facilitates a streamlined workflow for data loading, ROI identification, cell classification, fluorescence intensity calculation, spectral analysis, and report generation, supporting both manual and automated high-throughput analysis. This versatile platform enables the comprehensive analysis of large imaging datasets. In conclusion, the FluoAnalysis MATLAB toolbox provides a robust and versatile platform for the integrated analysis of calcium imaging and electrophysiological data, supporting diverse neuroscience research applications.

PurposeTwo-photon excitation fluorescence (2PEF) reveals information about tissue function. Concerns for phototoxicity demand lower light exposure during imaging. Reducing excitation light reduces the quality of the image by limiting fluorescence emission. We applied deep learning (DL) super-resolution techniques to images acquired from low light exposure to yield high-resolution images of retinal and skin tissues.MethodsWe analyzed two methods: a method based on U-Net and a patch-based regression method using paired images of skin (550) and retina (1200), each with low- and high-resolution paired images. The retina dataset was acquired at low and high laser powers from retinal organoids, and the skin dataset was obtained from averaging 7 to 15 frames or 70 frames. Mean squared error (MSE) and the structural similarity index measure (SSIM) were outcome measures for DL algorithm performance.ResultsFor the skin dataset, the patches method achieved a lower MSE (3.768) compared with U-Net (4.032) and a high SSIM (0.824) compared with U-Net (0.783). For the retinal dataset, the patches method achieved an average MSE of 27,611 compared with 146,855 for the U-Net method and an average SSIM of 0.636 compared with 0.607 for the U-Net method. The patches method was slower (303 seconds) than the U-Net method (

Background: In vivo two-photon imaging is a reliable method with high spatial resolution that allows observation of individual neuron and dendritic activity longitudinally. Neurons in local brain regions can be influenced by global brain states such as levels of arousal and attention that change over relatively short time scales, such as minutes. As such, the scientific rigor of investigating regional neuronal activities could be enhanced by considering the global brain state. New method: In order to assess the global brain state during in vivo two-photon imaging, CBRAIN (collective brain research platform aided by illuminating neural activity), a wireless EEG collecting and labeling device, was controlled by the same computer of two-photon microscope. In an experiment to explore neuronal responses to isoflurane anesthesia through two-photon imaging, we investigated whether the response of individual cells correlated with concurrent EEG changes induced by anesthesia. Results: In two-photon imaging, calcium activities of the excitatory neurons in the primary somatosensory cortex disappeared in about 30s after to the initiation of isoflurane anesthesia. The simultaneously recorded EEG showed various transitional activity for about 7 min from the initiation of anesthesia and continued with burst and suppression alternating pattern thereafter. As such, there was a dissociation between excitatory neuron activity of the primary somatosensory cortex and the global brain activity under anesthesia. Comparison with existing method(s): Existing methods to combine two-photon and EEG recording used wired EEG recording. In this study, wireless EEG was used in conjunction with two-photon imaging, facilitated by CBRAIN. More importantly, built-in algorithms of the CBRAIN can automatically detect brain state such as sleep. The codes used for EEG classification are easy to use, with no prior experience required. Conclusion: Simultaneous recording of wireless EEG and two-photon imaging provides a practical way to capture individual neuronal activities with respect to global brain state in an experimental set-up.

In recent years, the development of carbon dot‐based fluorescent nanoparticles for bioimaging applications has attracted the attention of the scientific community. However, many of these systems absorb and emit in the blue–green region of the electromagnetic spectrum, limiting their application in bioimaging. Herein, the facile design and development of highly efficient two‐photon excitable red‐emissive carbon dots are reported and their high performance in bioimaging applications is demonstrated. The importance of aromatic precursors in developing red‐emissive carbon dots is demonstrated. The optimized carbon dots are highly biocompatible and nontoxic, with remarkable photostability in cells under two‐photon near‐infrared excitation. The present study points to the great potential of two‐photon excitable red‐emissive carbon dot as an efficient bioimaging agent for cellular biolabeling, long‐term and real‐time cellular imaging, and high‐resolution deep‐tissue imaging in complex biological systems.

Antibiotics are emerging contaminants in the environment, and their monitoring is important for food safety and human health. Herein, we synthesized a two-photon carbon quantum dots (TP-CQDs) nanoprobe using a simple and green hydrothermal approach and applied it as a chemosensor to detect chlortetracycline (CTC). CTC quenches the intrinsic fluorescence of TP-CQDs, providing a linear response within the 0.5 to 60 μM range and yielding a limit of detection of only 25 nM. Besides excellent stability and good optical properties, we found that TP-CQDs are selective to CTC as structurally similar antibiotics, along with cations, anions, and amino acids, had no significant influence on fluorescence emission. Finally, we successfully applied this sensing platform to quantify CTC in milk, fish, and eggs. [ABSTRACT FROM AUTHOR]

AMPA receptors (AMPARs) play a critical role in synaptic plasticity and learning and memory, and dysfunction or dysregulation of AMPARs could lead to various neurological and psychiatric disorders, such as Alzheimer's disease (AD). However, the dynamics and/or longitudinal changes of AMPARs in vivo during AD pathogenesis remain elusive. Here, employing 5xFAD SEP-GluA1 KI mice, we investigated endogenous AMPA receptor dynamics in a whisker deflection-associated Go/No-go learning paradigm. We found a significant increase in synaptosomal AMPA receptor subunits GluA1 in WT mice after learning, while no such changes were detected in 7-mo-old 5xFAD mice. Daily training led to an increase in endogenous spine surface GluA1 in Control mice, while this increase was absent in 5xFAD-KI mice which correlates with its learning defects in Go/No-go paradigm. Furthermore, we demonstrated that the onset of abnormal AMPAR dynamics corresponds temporally with microglia and astrocyte overactivation. Our results have shown that impairments in endogenous AMPA receptor dynamics play an important role in learning deficits in 5xFAD mice and AD pathogenesis. [ABSTRACT FROM AUTHOR]

Multi‐color two‐photon microscopy imaging of live cells is essential in biology. However, the limited diffraction resolution of conventional two‐photon microscopy restricts its application to subcellular organelle imaging. Recently, we developed a laser scanning two‐photon non‐linear structured illumination microscope (2P‐NLSIM), whose resolution improved three‐fold. However, its ability to image polychromatic live cells under low excitation power has not been verified. Here, to improve the reconstruction super‐resolution image quality under low excitation power, we increased the image modulation depth by multiplying the raw images with the reference fringe patterns in the reconstruction process. Simultaneously, we optimized the 2P‐NLSIM system to image live cells, including the excitation power, imaging speed, and field of view. The proposed system could provide a new imaging tool for live cells. [ABSTRACT FROM AUTHOR]

The axon initial segment (AIS) is a specialized neuronal compartment in which synaptic input is converted into action potential (AP) output. This process is supported by a diverse complement of sodium, potassium, and calcium channels (CaV). Different classes of sodium and potassium channels are scaffolded at specific sites within the AIS, conferring unique functions, but how calcium channels are functionally distributed within the AIS is unclear. Here, we use conventional two-photon laser scanning and diffraction-limited, high-speed spot two-photon imaging to resolve AP-evoked calcium dynamics in the AIS with high spatiotemporal resolution. In mouse layer 5 prefrontal pyramidal neurons, calcium influx was mediated by a mix of CaV2 and CaV3 channels that differentially localized to discrete regions. CaV3 functionally localized to produce nanodomain hotspots of calcium influx that coupled to ryanodine-sensitive stores, whereas CaV2 localized to non-hotspot regions. Thus, different pools of CaVs appear to play distinct roles in AIS function.SIGNIFICANCE STATEMENT The axon initial segment (AIS) is the site where synaptic input is transformed into action potential (AP) output. It achieves this function through a diverse complement of sodium, potassium, and calcium channels (CaV). While the localization and function of sodium channels and potassium channels at the AIS is well described, less is known about the functional distribution of CaVs. We used high-speed two-photon imaging to understand activity-dependent calcium dynamics in the AIS of mouse neocortical pyramidal neurons. Surprisingly, we found that calcium influx occurred in two distinct domains: CaV3 generates hotspot regions of calcium influx coupled to calcium stores, whereas CaV2 channels underlie diffuse calcium influx between hotspots. Therefore, different CaV classes localize to distinct AIS subdomains, possibly regulating distinct cellular processes.

There has been considerable research showing populations of neurons encoding for different aspects of space in the brain. Recently, several studies using two-photon calcium imaging and virtual navigation have identified "spatially" modulated neurons in the posterior cortex. We enquire here whether the presence of such spatial representations may be a cortex-wide phenomenon and, if so, whether these representations can be organized in the absence of the hippocampus. To this end, we imaged the dorsal cortex of mice running on a treadmill populated with tactile cues. A high percentage (40-80%) of the detected neurons exhibited sparse, spatially localized activity, with activity fields uniformly localized over the track. The development of this location specificity was impaired by hippocampal damage. Thus, there is a substantial population of neurons distributed widely over the cortex that collectively form a continuous representation of the explored environment, and hippocampal outflow is necessary to organize this phenomenon.SIGNIFICANCE STATEMENT Increasing evidence points to the role of the neocortex in encoding spatial information. Whether this feature is linked to hippocampal functions is largely unknown. Here, we systematically surveyed multiple regions in the dorsal cortex of the same animal for the presence of signals encoding for spatial position. We described populations of cortical neurons expressing sequential patterns of activity localized in space in primary, secondary, and associational areas. Furthermore, we showed that the formation of these spatial representations was impacted by hippocampal lesion. Our results indicate that hippocampal inputs are necessary to maintain a precise cortical representation of space.

A titled tetraphenylethene imidazole compound with methoxy group named 4, 5-bis (4-methoxyphenyl) -2- (4- (1, 2, 2-triphenylvinyl) phenyl) -1H-imidazole (BTI) is synthesized by one step. The target BTI is fully characterized by NMR spectroscopy, mass spectrometry and elemental analysis. The single crystal structure of BTI is obtained by evaporation of the mother solutions. The photophysical properties of BTI are studied by UVVis absorption and fluorescence emission spectrum. By means of theoretical calculation, the excited state properties of BTI are elaborated. Finally, the application of BTI in cell imaging is studied. The results show that BTI exhibits typical aggregation-induced emission (AIE) properties with intramolecular charge transfer (ICT) character, and the experimental spectral data agree well with the theoretical calculation results. The cell imaging study shows that BTI can well penetrate the HepG2 cell membrane and perform one-and twophoton excitated confocal fluorescence imaging. [ABSTRACT FROM AUTHOR]

Functional nucleic acid (FNA) probes have been widely used in environmental monitoring, food analysis, clinical diagnosis, and biological imaging because of their easy synthesis, functional modification, flexible design, and stable properties. However, most FNA probes are designed based on one-photon (OP) in the ultraviolet or visible regions, and the effectiveness of these OP-based FNA probes may be hindered by certain factors, such as their potential for photodamage and limited light tissue penetration. Two-photon (TP) is characterized by the nonlinear absorption of two relatively low-energy photons of near-infrared (NIR) light with the resulting emission of high-energy ultraviolet or visible light. TP-based FNA probes have excellent properties, including lower tissue self-absorption and autofluorescence, reduced photodamage and photobleaching, and higher spatial resolution, making them more advantageous than the conventional OP-based FNA probes in biomedical sensing. In this review, we summarize the recent advances of TP-excited and -activated FNA probes and detail their applications in biomolecular detection. In addition, we also share our views on the highlights and limitations of TP-based FNA probes. The ultimate goal is to provide design approaches for the development of high-performance TP-based FNA probes, thereby promoting their biological applications. [ABSTRACT FROM AUTHOR]

Multiphoton absorption of entangled photons offers ways for obtaining unique information about chemical and biological processes. Measurements with entangled photons may enable sensing biological signatures with high selectivity and at very low light levels to protect against photodamage. In this paper, we present a theoretical and experimental study of the excitation wavelength dependence of the entangled two-photon absorption (ETPA) process in a molecular system, which provides insights into how entanglement affects molecular spectra. We demonstrate that the ETPA excitation spectrum can be different from that of classical TPA as well as that for one-photon resonant absorption (OPA) with photons of doubled frequency. These results are modeled by assuming the ETPA cross-section is governed by a two-photon excited state radiative linewidth rather than by electron-phonon interactions, and this leads to excitation spectra that match the observed results. Further, we find that the two-photon-allowed states with highest TPA and ETPA intensities have high electronic entanglements, with ETPA especially favoring states with the longest radiative lifetimes. These results provide concepts for the development of quantum light--based spectroscopy and microscopy that will lead to much higher efficiency of ETPA sensors and low- intensity detection schemes. [ABSTRACT FROM AUTHOR]

High-resolution imaging techniques capable of detecting identifiable endogenous fluorophores in the eye along with genetic testing will dramatically improve diagnostic capabilities in the ophthalmology clinic and accelerate the development of new treatments for blinding diseases. Two-photon excitation (TPE)-based imaging overcomes the filtering of ultraviolet light by the lens of the human eye and thus can be utilized to discover defects in vitamin A metabolism during the regeneration of the visual pigments required for the detection of light. Combining TPE with fluorescence lifetime imaging (FLIM) and spectral analyses offers the potential of detecting diseases of the retina at earlier stages before irreversible structural damage has occurred. The main barriers to realizing the benefits of TPE for imaging the human retina arise from concerns about the high light exposure typically needed for informative TPE imaging and the requirement to correlate the ensuing data with different states of health and disease. To overcome these hurdles, we improved TPE efficiency by controlling temporal properties of the excitation light and employed phasor analyses to FLIM and spectral data in mouse models of retinal diseases. Modeling of retinal photodamage revealed that plasma-mediated effects do not play a role and that melanin-related thermal effects are mitigated by reducing pulse repetition frequency. By using noninvasive TPE imaging we identified molecular components of individual granules in the retinal pigment epithelium and present their analytical characteristics.

T lymphocyte motility and interaction dynamics with other immune cells are vital determinants of immune responses. Regulatory T (Treg) cells prevent autoimmune disorders by suppressing excessive lymphocyte activity, but how interstitial motility patterns of Treg cells limit neuroinflammation is not well understood. We used two-photon microscopy to elucidate the spatial organization, motility characteristics, and interactions of endogenous Treg and Th17 cells together with antigen-presenting cells (APCs) within the spinal cord leptomeninges in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Th17 cells arrive before the onset of clinical symptoms, distribute uniformly during the peak, and decline in numbers during later stages of EAE. In contrast, Treg cells arrive after Th17 cells and persist during the chronic phase. Th17 cells meander widely, interact with APCs, and exhibit cytosolic Ca2+ transients and elevated basal Ca2+ levels before the arrival of Treg cells. In contrast, Treg cells adopt a confined, repetitive-scanning motility while contacting APCs. These locally confined but highly motile Treg cells limit Th17 cells from accessing APCs and suppress Th17 cell Ca2+ signaling by a mechanism that is upstream of store-operated Ca2+ entry. Finally, Treg cell depletion increases APC numbers in the spinal cord and exaggerates ongoing neuroinflammation. Our results point to fundamental differences in motility characteristics between Th17 and Treg cells in the inflamed spinal cord and reveal three potential cellular mechanisms by which Treg cells regulate Th17 cell effector functions: reduction of APC density, limiting access of Th17 cells to APCs, and suppression of Th17 Ca2+ signaling.

Fluorescence calcium imaging using a range of microscopy approaches, such as two-photon excitation or head-mounted miniscopes, is one of the preferred methods to record neuronal activity and glial signals in various experimental settings, including acute brain slices, brain organoids, and behaving animals. Because changes in the fluorescence intensity of genetically encoded or chemical calcium indicators correlate with action potential firing in neurons, data analysis is based on inferring such spiking from changes in pixel intensity values across time within different regions of interest. However, the algorithms necessary to extract biologically relevant information from these fluorescent signals are complex and require significant expertise in programming to develop robust analysis pipelines. For decades, the only way to perform these analyses was for individual laboratories to write their custom code. These routines were typically not well annotated and lacked intuitive graphical user interfaces (GUIs), which made it difficult for scientists in other laboratories to adopt them. Although the panorama is changing with recent tools like CaImAn, Suite2P, and others, there is still a barrier for many laboratories to adopt these packages, especially for potential users without sophisticated programming skills. As two-photon microscopes are becoming increasingly affordable, the bottleneck is no longer the hardware, but the software used to analyze the calcium data optimally and consistently across different groups. We addressed this unmet need by incorporating recent software solutions, namely NoRMCorre and CaImAn, for motion correction, segmentation, signal extraction, and deconvolution of calcium imaging data into an open-source, easy to use, GUI-based, intuitive and automated data analysis software package, which we named EZcalcium.

Fluorescence calcium imaging using a range of microscopy approaches, such as two-photon excitation or head-mounted "miniscopes," is one of the preferred methods to record neuronal activity and glial signals in various experimental settings, including acute brain slices, brain organoids, and behaving animals. Because changes in the fluorescence intensity of genetically encoded or chemical calcium indicators correlate with action potential firing in neurons, data analysis is based on inferring such spiking from changes in pixel intensity values across time within different regions of interest. However, the algorithms necessary to extract biologically relevant information from these fluorescent signals are complex and require significant expertise in programming to develop robust analysis pipelines. For decades, the only way to perform these analyses was for individual laboratories to write their custom code. These routines were typically not well annotated and lacked intuitive graphical user interfaces (GUIs), which made it difficult for scientists in other laboratories to adopt them. Although the panorama is changing with recent tools like CaImAn, Suite2P, and others, there is still a barrier for many laboratories to adopt these packages, especially for potential users without sophisticated programming skills. As two-photon microscopes are becoming increasingly affordable, the bottleneck is no longer the hardware, but the software used to analyze the calcium data optimally and consistently across different groups. We addressed this unmet need by incorporating recent software solutions, namely NoRMCorre and CaImAn, for motion correction, segmentation, signal extraction, and deconvolution of calcium imaging data into an open-source, easy to use, GUI-based, intuitive and automated data analysis software package, which we named EZcalcium.

This report details spectroscopic characterizations of rare-earth, core-shell nanoparticles decorated with the f-element chelator 3,4,3-LI(1,2-HOPO). Evidence of photon downconversion is corroborated through detailed power dependence measurements, which suggest two-photon decay paths are active in these materials, albeit only representing a minority contribution of the sum luminescence, with emission being dominated by normal, Stokes' shifted fluorescence. Specifically, ultraviolet ligand photosensitization of Nd3+ ions in a NaGdF4 host shell results in energy transfer to a Nd3+/Yb3+-doped NaGdF4 nanoparticle core. The population and subsequent decay of core, Yb3+ 2 F 5/2 states result in a spectral shift of 620 nm, manifested in a NIR emission displaying luminescence profiles diagnostic of Yb3+ and Nd3+ excited state decays. Emphasis is placed on the generality of this material architecture for realizing ligand-pumped, multi-photon downconversion, with the Nd3+/Yb3+ system presented here functioning as a working prototype for a design principle that may be readily extended to other lanthanide pairs.

Fluorescent probe is an important tool for investigation of biological events in plant tissues. However, the research on small-molecule based fluorescent probe for plant imaging still stays at the initial stage. In order to noninvasively achieve the subcellular information of Arabidopsis thaliana, a two-photon and fluorene based fluorophore with a bi-oligo (ethylene glycol) functional group was synthesized. Under the laser's irradiation at 750 nm, the subcellular structures of Arabidopsis thaliana including stomata and root tips were clearly observed without slicing, which displayed significant superiorities over traditional single-photon excitation microscopy. Subsequently, the above functionalized fluorophore was further modified with a recognition group (dipicolylamine) to form a smart fluorescent probe (LJTP1). As results, LJTP1 not only can sensitively detect Cu2+/S2− with fluorescence “on-off-on” response in vitro and in vivo, but also can achieve noninvasive imaging in Arabidopsis thaliana tissues under two-photon microscopy. We believe this study will open up a new mind for noninvasive imaging in plant tissues.

To understand how neural activity encodes and coordinates behavior, it is desirable to record multi-neuronal activity in freely behaving animals. Imaging in unrestrained animals is challenging, especially for those, like larval Drosophila melanogaster, whose brains are deformed by body motion. A previously demonstrated two-photon trackingmicroscope recorded fromindividual neurons in freely crawling Drosophila larvae but faced limits in multi-neuronal recording. Here we demonstrate a new tracking microscope using acousto-optic deflectors (AODs) and an acoustic GRIN lens (TAG lens) to achieve axially resonant 2D randomaccess scanning, sampling along arbitrarily located axial lines at a line rate of 70 kHz. With a tracking latency of 0.1 ms, this microscope recorded activities of various neurons in moving larval Drosophila CNS and VNC including premotor neurons, bilateral visual interneurons, and descending command neurons. This technique can be applied to the existing two-photon microscope to allow for fast 3D tracking and scanning. [ABSTRACT FROM AUTHOR]

Different segments of the cerebral vascular network may react distinctly to brain ischemia and recanalization. However, there are limited systematic observations of these vascular responses in mice under a physiological state following ischemic stroke. Herein, we aimed to investigate the vasodynamics among several segments along the cerebral vessels in awake mice following cerebral ischemia/recanalization via two-photon imaging. Plasma in the blood vessels were labelled with fluorescein isothiocyanate dextran. Smooth muscle cells and pericytes were labelled via a genetic mouse line (PDGFRβ-tdTomato). We observed a no-reflow phenomenon in downstream microcirculation, and the vasodynamics of different segments of larger cerebral vessels varied in the penumbra area following cerebral ischemia-reperfusion. Despite obtaining reperfusion from the middle cerebral artery, there were significant constrictions of the downstream blood vessels in the ischemic penumbra zone. Interestingly, we observed an extensive constriction of the capillaries 3 hours following recanalization, both at the site covered by pericyte soma and by the pericyte process alone. In addition, we did not observe a significant positive correlation between the changed capillary diameter and pericyte coverage along the capillary. Taken together, abnormal constrictions and vasodynamics of cerebral large and small vessels may directly contribute to microcirculation failure following recanalization in ischemic stroke. [ABSTRACT FROM AUTHOR]

Cytochrome P450 1A is one of the vital subfamilies of heme-containing cytochrome P450 enzymes belonging to an important exogenous metabolizing CYP in human. The abnormal of endoplasmic reticulum (ER) may directly affect the functional activity of ER-located CYP1A and be associated with the occurrence and development of various diseases. In the present study, we constructed a selective two-photon fluorescent probe ERNM for rapid and visual detection of endogenous CYP1A that was localized in the ER. ERNM could target the ER and detect the enzymatically active CYP1A in living cells and tissues. The monitoring ability of ERNM for the fluctuations in functionality level of CYP1A was confirmed using ER stressed A549 cell. Based on the ER-targeting two-photon probe for CYP1A, the close association of ER state and the functional activity of ER-locating CYP1A was confirmed, which would promote the deep understanding of the biofunction of CYP1A in various ER-related diseases. [ABSTRACT FROM AUTHOR]

Our understanding of molecules has stagnated at a single quantum system, with atoms as Newtonian particles and electrons as quantum particles. Here, however, we reveal that both atoms and electrons in a molecule are quantum particles, and their quantum–quantum interactions create a previously unknown, newfangled molecular property—supracence. Molecular supracence is a phenomenon in which the molecule transfers its potential energy from quantum atoms to photo-excited electrons so that the emitted photon has more energy than that of the absorbed one. Importantly, experiments reveal such quantum energy exchanges are independent of temperature. When quantum fluctuation results in absorbing low-energy photons, yet still emitting high-energy photons, supracence occurs. This report, therefore, reveals novel principles governing molecular supracence via experiments that were rationalized by full quantum (FQ) theory. This advancement in understanding predicts the super-spectral resolution of supracence, and molecular imaging confirms such innovative forecasts using closely emitting rhodamine 123 and rhodamine B in living cell imaging of mitochondria and endosomes. [ABSTRACT FROM AUTHOR]

Understanding the functions of enzymes in various physiological processes is important, but the design of signaling probes for fast analysis of enzymatic activity is particularly challenging. Herein, a fluorescence-enhanced probe, 10-methyl-2-nitro-acridone (MNA), was synthesized and applied to analyze nitroreductase (NTR) activity in vitro and in vivo. The detection mechanism is based on the nitro group in MNA reacting toward NTR with high reactivity and generating 10-methyl-2-amino- acridone (MAA) accompanied by an obvious fluorescence signal enhancement at 525 nm emission. The probe shows low cytotoxicity, fast response, and high selectivity and sensitivity with a limit of detection as low as 150 ng·mL−1. The probe was also employed for two-photon fluorescence imaging of NTR in zebrafish in vivo revealing the distribution of NTR. Versus existing NTR probes, the proposed probe shows favorable analytical performance including near-infrared light excitation with no other byproducts produced after the reaction. The superior properties of this signaling probe allow it to become a fluorescence imaging candidate in other biosystems.

Decision making is often driven by the subjective value of available options, a value which is formed through experience. To support this fundamental behavior, the brain must encode and maintain the subjective value. To investigate the area specificity and plasticity of value coding, we trained mice in a value-based decision task and imaged neural activity in 6 cortical areas with cellular resolution. History- and value-related signals were widespread across areas, but their strength and temporal patterns differed. In expert mice, the retrosplenial cortex (RSC) uniquely encoded history- and value-related signals with persistent population activity patterns across trials. This unique encoding of RSC emerged during task learning with a strong increase in more distant history signals. Acute inactivation of RSC selectively impaired the reward-history-based behavioral strategy. Our results indicate that RSC flexibly changes its history coding and persistently encodes value-related signals to support adaptive behaviors.

To understand how neural activity encodes and coordinates behavior, it is desirable to record multi-neuronal activity in freely behaving animals. Imaging in unrestrained animals is challenging, especially for those, like larval Drosophila melanogaster, whose brains are deformed by body motion. A previously demonstrated two-photon tracking microscope recorded from individual neurons in freely crawling Drosophila larvae but faced limits in multi-neuronal recording. Here we demonstrate a new tracking microscope using acousto-optic deflectors (AODs) and an acoustic GRIN lens (TAG lens) to achieve axially resonant 2D random access scanning, sampling along arbitrarily located axial lines at a line rate of 70 kHz. With a tracking latency of 0.1 ms, this microscope recorded activities of various neurons in moving larval Drosophila CNS and VNC including premotor neurons, bilateral visual interneurons, and descending command neurons. This technique can be applied to the existing two-photon microscope to allow for fast 3D tracking and scanning.

Multifocus gratings (MFGs) enable microscopes and other imaging systems to record entire Z-stacks of images in a single camera exposure. The exact grating shape depends on microscope parameters like wavelength and magnification and defines the multiplexing onto a grid of MxN Z-slices. To facilitate the swift production and alteration of MFGs for a system and application at hand, we have developed a fabrication protocol that allows manufacturing of 1xN MFGs within hours and without the requirement of clean room facilities or hazardous etching steps. Our approach uses photolithography with a custom-built stage-scanning direct laser writing (DLW) system. By writing MFG grating lines into spin-coated negative tone SU-8 photoresist, polymerized parts are crafted onto the substrate and thus directly become a part of the grating structure. We provide software to generate the required MFG grating line paths, details of the DLW system and fully characterize a manufactured MFG. Our produced MFG is 5.4 mm in diameter and manages to record an image volume with a Z-span of over 600 μm without spherical aberrations or noticeable loss of resolution.

In recent years, the development of novel photocrosslinking strategies and photoactivatable materials has stimulated widespread use of light-mediated biofabrication techniques. However, despite great progress toward more efficient and biocompatible photochemical strategies, current photoresins still rely on photoinitiators (PIs) producing radical-initiating species to trigger the so-called free-radical crosslinking/polymerization. In the context of bioprinting, where cells are encapsulated in the bioink, the presence of radicals raises concerns of potential cytotoxicity. In this work, a universal, radical-free (RF) photocrosslinking strategy to be used for light-based technologies is presented. Leveraging RF uncaging mechanisms and Michael addition, cell-laden constructs are photocrosslinked by means of one- and two-photon excitation with high biocompatibility. A hydrophilic coumarin-based group is used to cage a universal RF photocrosslinker based on 4-arm-PEG-thiol (PEG4SH). Upon light exposure, thiols are uncaged and react with an alkene counterpart to form a hydrogel. RF photocrosslinker is shown to be highly stable, enabling potential for off-the-shelf products. While PI-based systems cause a strong upregulation of reactive oxygen species (ROS)-associated genes, ROS are not detected in RF photoresins. Finally, optimized RF photoresin is successfully exploited for high resolution two-photon stereolithography (2P-SL) using remarkably low polymer concentration (<1.5%), paving the way for a shift toward radical-free light-based bioprinting. [ABSTRACT FROM AUTHOR]

Adoptive transfer of T-cells expressing chimeric antigen receptors (CAR) has shown remarkable clinical efficacy against advanced B-cell malignancies. Nonetheless, the field of CAR T-cells is currently facing several major challenges. In particular, the CAR T-cell strategy has not yet produced favorable clinical responses when targeting solid tumors. In this context, it is of paramount importance to understand the determinants that limit the efficacy of T-cell-based immunotherapy. Characterization of CAR T-cells is usually based on flow cytometry and whole-transcriptome profiling. These approaches have been very valuable to determine intrinsic elements that condition T-cell ability to proliferate and expand. However, they do not take into account spatial and kinetic aspects of T-cell responses. In particular, in order to control tumor growth, CAR T-cells need to enter into the tumor, migrate within a complex tumor environment, and form productive conjugates with their targets. Advanced imaging techniques combined with innovative preclinical models represent promising tools to uncover the dynamics of CAR T-cells. In this review, we will discuss recent results on the biology of engineered T-cells that have been obtained with real-time imaging microscopy. Important notions have emerged from these imaging-based studies, such as the multi-killing potential of CAR T-cells. Finally, we will highlight how imaging techniques combined with other tools can solve remaining unresolved questions in the field of engineered T-cells. [ABSTRACT FROM AUTHOR]